The invention relates to acetyl coenzyme A carboxylase genes of the fungus Aspergillus fumigatus and their use in identifying antifungal agents.
The enzyme acetyl coenzyme A carboxylase (ACCase) is responsible for synthesizing malonyl CoA from acetyl CoA. ACCase is essential for synthesis of fatty acids.
By way of background, the Fungi Kingdom consists of two divisions, the Eumycota and Myxomycota or the true fungi and slime molds, respectively. The true fungi are those species that are hyphal or are clearly related to species that are hyphal, possess cell walls throughout most or all of their life cycle, and are exclusively absorptive in their function. The slime molds are organisms that do not form hyphae, lack cell walls during the phase in which they obtain nutrients and grow and are capable of ingesting nutrients in particulate form by phagocytosis.
The two most important classes of true fungi in which most species produce motile cells, known as zoospores, are the Oomycetes, and the Chytridiomycetes. The fungi that lack zoospores are classified according to the sexual phase of the fungal life cycle. The sexual process leads to the production of characteristic spores in the different groups. The fungi that form zygospores are classified as Zygomycetes, those that form ascospores are classified as Ascomycetes, and those forming basidiospores are classified as Basidiomycetes. There are also many species, recognizable as higher fungi through the presence of cell walls in their hyphae, that produce asexual spores but lack a sexual phase. These are known as Deuteromycetes, and details of their asexual sporulation are used to classify them. A representative member of the Deuteromycetes includes Candida albicans. These species are extensively reviewed in xe2x80x9cThe Fungixe2x80x9d (Ed M J Carlile and S C Watkinson 1994 Acad Press Ltd) and xe2x80x9cThe Growing Fungusxe2x80x9d (Ed. N A R Gow and G M Gadd, 1995, Chapman and Hall).
Yeast are fungi that are normally unicellular and reproduce by budding although some will, under appropriate conditions, produce hyphae, just as some normally hyphal fungi may produce a yeast phase. The best known of all yeasts is Saccharomyces cerevisiae, which is a member of the Ascomycetes species. It is commonly regarded as a diploid yeast since mating usually soon follows ascospore germination. However, single cells can be used to establish permanently haploid cultures.
Fungal and other mycotic pathogens are responsible for a variety of diseases in humans, animals and plants. Fungal infection is also a significant problem in veterinary medicine. Some of the fungi that infect animals can be transmitted from animals to humans. Fungal infections or infestations are also a very serious problem in agriculture with fungicides being employed to protect vegetable and fruit and cereal crops. Fungal attack of wood products is also of major economic importance. Additional products that are susceptible to fungal infestation include textiles, plastics, paper and paint. Some of these fungal targets are extensively reviewed in WO 95/11969.
Statistics show that the incidence of fungal infections has doubled from the 1980""s to the 1990""s, and infections of the blood stream have increased fivefold with an observed mortality of 50% (Tally et al., 1997, Int. Conference Biotechnol Microb. Prods: Novel Pharmacol. Agrobiol. Activities, Williamsburg, Va. Abstr S5 p19). These include those fungal infections, such as candidiasis, to which all individuals are susceptible, but also infections such as cryptococcosis and aspergillosis, which occur particularly in patients of compromised immune status.
By way of example, the yeast Candida albicans (C. albicans) has been shown to be one of the most pervasive fungal pathogens in humans. It has the capacity to opportunistically infect a diverse spectrum of compromised hosts, and to invade many diverse tissues in the human body. It can in many instances evade antibiotic treatment and the immune system. Although Candida albicans is a member of the normal flora of the mucous membranes in the respiratory, gastrointestinal, and female genital tracts, in such locations, it may gain dominance and be associated with pathologic conditions. Sometimes it produces progressive systematic disease in debilitated or immunosuppressed patients, particularly if cell-mediated immunity is impaired. Sepsis may occur in patients with compromised cellular immunity, e.g., those undergoing cancer chemotherapy or those with lymphoma, AIDS, or other conditions. Candida may produce bloodstream invasion, thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue when introduced intravenously, e.g., via tubing, needles, narcotics abuse etc.
Candida albicans has been shown to be diploid with balanced lethals, and therefore probably does not go through a sexual phase or meiotic cycle. This yeast appears to be able to spontaneously and reversibly switch at high frequency between at least seven general phenotypes. Switching has been shown to occur not only in standard laboratory strains, but also in strains isolated from the mouths of healthy individuals.
Nystatin, ketoconazole, and amphotericin B are drugs that have been used to treat oral and systemic Candida infections. However, orally administered nyastin is limited to treatment within the gut and is not applicable to systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these drugs may not be effective in such treatment unless combined with additional drugs. Amphotericin B has a relatively narrow therapeutic index and numerous undesirable side effects, ranging from nausea and vomiting to kidney damage and toxicities occur even at therapeutic concentrations. While ketoconazole and other azole antifungals exhibit significantly lower toxicity, their mechanism of action, through inactivation of cytochrome P450 prosthetic group in certain enzymes (some of which are found in humans) precludes use in patients that are simultaneously receiving other drugs that are metabolized by the body""s cytochrome P450 enzymes. These adverse effects mean that their use is generally limited to the treatment of topical or superficial infections. In addition, resistance to these compounds is emerging and may pose a serious problem in the future. The more recently developed triazole drugs, such as fluconazole, are believed by some to have fewer side effects but are not completely effective against all pathogens.
Invasive aspergillosis, caused by Aspergillus fumigatus (A. fumigatus) has also become an increasingly opportunistic infection. There has been a 14-fold increase in its incidence during the past 12 years as detected by autopsy, and only two drugs are available that are effective in its treatment, neither of which is completely satisfactory. Amphotericin B needs to be given intravenously and has a number of toxic side effects. Itraconazole, which can be given orally is often prescribed imprudently, encouraging the emergence of resistant fungal strains (Dunn-Coleman and Prade, Nature Biotechnology, 1998, 16: 5). Resistance is also developing to synthetic azoles (such as fluconazole and flucytosine), and the natural polyenes (such as amphotericin B) are limited in use by their toxicity.
Fungicide resistance generally develops when a fungal cell or fungal population that originally was sensitive to a fungicide becomes less sensitive by heritable changes after a period of exposure to the fungicide.
In certain applications, such as agriculture, it is possible to combat resistance through alteration of fungicides or the use of fungicide mixtures. To prevent or delay the build up of a resistant pathogen population, different agents that are effective against a particular disease must be available. One way of increasing the number of available agents is to search for new site-specific inhibitors.
Consequently, antifungal drug discovery efforts have been directed at components of the fungal cell or its metabolism that are unique to fungi, and hence might be used as therapeutic targets of new agents which act on the fungal pathogen without undue toxicity to host cells. Such potential targets include enzymes critical to fungal cell wall assembly (U.S. Pat. No. 5,194,600) as well as topoisomerases (enzymes required for replication of fungal DNA). Two semisynthetic antifungal agents such as the echinocandins and the related pneumocandins are in late stage clinical trials. Both are cyclic lipopeptides produced by fungi that non-competitively inhibit (1,3)-glucan synthase and thus interfere with the biosynthesis of the fungal cell wall. These clinical candidates are generally more water-soluble, have improved pharmacokinetics and broader antifungal spectra than their natural parent compounds and have activity spectra that include many Candida species, including Candida albicans, and Aspergilli.
Because no single approach may be effective against all fungal pathogens, however, and because of the possibility of developed resistance to previously effective antifungal compounds, there remains a need for new antifungal agents with novel mechanisms of action and improved or different activity profiles. There is also a need for agents which are active against fungi but are not toxic to mammalian cells, as toxicity to mammalian cells can lead to a low therapeutic index and undesirable side effects in the host (e.g., patient). An important aspect of meeting this need is the selection of an appropriate component of fungal structure or metabolism as a therapeutic target.
Even after a particular intracellular target is selected, the means by which new antifungal agents are identified pose certain challenges. Despite the increased use of rational drug design, a preferred method continues to be the mass screening of compound xe2x80x9clibrariesxe2x80x9d for active agents by exposing cultures of fungal pathogens to the test compounds and assaying for inhibition of growth. In testing thousands or tens of thousands of compounds, however, a correspondingly large number of fungal cultures must be grown over time periods which are relatively long compared to most bacterial culture times. Moreover, a compound which is found to inhibit fungal growth in culture may be acting not on the desired target but on a different, less unique fungal component, with the result that the compound may act against host cells as well and thereby produce unacceptable side effects. Consequently, there is a need for an assay or screening methods which more specifically identifies those agents that are active against a certain intracellular target. Additionally, there is a need for assay methods having greater throughput, that is, assay methods which reduce the time and materials needed to test each compound of interest.
Although cyclic lipopeptides produced by fungi are in late-stage clinical trials as potential anti-fungal agents, the lipid biosynthesis and degradation pathways have been only sparingly investigated in fungi. This area is reviewed by Weete (1980 Lipid Biochemistry of Fungi and Other Organisms, Plenum New York) and Chopra and Khuller (1984 Crit Rev Microbiol 11: 209-250). It is known that fungal biosynthesis of fatty acids takes place in the cytosol and starts with carboxylation of acetyl-CoA to malonyl-CoA. From this malonyl-CoA consecutive C2 units are added to acetyl-CoA or the growing fatty-CoA ester chain by a complex fatty acid synthase complex harboring seven different enzymatic activities. In contrast, knowledge of lipid biosynthesis and degradation has come from research in other organisms.
By way of example, it has been recognized that the biosynthesis of very long chain fatty acids in organisms other than fungi requires four enzyme systems: acetyl coenzyme A (CoA) carboxylase, fatty acid synthetase, fatty acid desaturase, and fatty acyl chain elongation system. The rate limiting step of the de novo synthesis of fatty acids is under the control of the first of these, acetyl-CoA carboxylase (EC 6.4.1.2). This enzyme catalyses the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA which serves as the two carbon unit donor for the subsequent synthesis of long chain fatty acids by the fatty acid synthase complex. The chain length of newly synthesized fatty acids appears to depend on the concentration of malonyl-CoA rather than on the activity of the fatty acid synthase complex. Acetyl-CoA carboxylase thus regulates both the overall rate of de novo synthesis and chain length distribution of long chain fatty acids.
Acetyl-CoA carboxylase has been isolated from chicken liver (Buckner and Kolattakudy 1976 Biochem 15: 1948-1957; Manning et al 1976 Biochem J 153: 463-468; Ahmad et al 1978 J Biol Chem 253: 1733-1737; Hardie and Cohen 1978 FEBS Lett 91: 1-7); rat heart (Thampy 1989 J Biol Chem 264: 17631-17634); brown and white adipose tissue (Bianchi et al 1990 J Biol Chem 265: 1502-1509; Iverson et al 1990 Biochem J 269: 365-371); chick embryo brain (Thampy and Koshy 1991 J Lipid Res 32: 1667-1673) and has been observed by immunological techniques in rat diaphragm muscle (Bianchi et al 1990 ibid). Acetyl-CoA carboxylase has also been found in human skeletal muscle and adipose tissue (Witters et al 1994 Int J Biochem 26: 589-594) and in rat skeletal muscle (Trumble et al 1991 Life Sci 49: 39-43).
Data have been accumulating from several laboratories characterizing the different isoforms of Acetyl-CoA carboxylase. Thampy (1989 ibid) and Bianchi et al (1990 ibid) have reported a molecular mass of 280 kDa for the Acetyl-CoA carboxylases from rat heart and diaphragm muscle respectively. More recently, two isoforms (HACC275 and HACC 265) have been identified in human tissue. The HACC 275 form is predominant in human skeletal muscle (Witters et al 1994 ibid). The rat skeletal muscle isoform appears to be similar in molecular mass to the HACC 275 form in humans. However, it has been recognized that until the Acetyl-CoA carboxylase gene(s) from each tissue are cloned and the mRNA species are characterized, assumption of equivalency of isoforms with molecular masses is conjectural (Trumble et al 1995 Eur J Biochem 231: 192-198).
Preliminary studies on Acetyl-CoA carboxylase (Acc1p) from yeast Saccharomyces has been shown to: (i) have a subunit molecular mass of 250 kDa, (ii) be active as a tetramer and (iii) be subject to short term regulation by phosphorylation (Al-Feel et al 1992 Proc Natl Acad Sci 89: 4534-4538; Obernayer and Lynen 1976: Trends Biochem Sci 1: 169-171; Witters et al 1990 Biochem Biophys Res Commun 169: 369-376). Genetic and biochemical analyses of fatty acid synthesis mutants and a conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1, have also indicated that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is an essential function of the acetyl-CoA carboxylase (ACC1) gene (Schneiter et al 1996 Mol and Cell Biol 16: 7161-7172).
The invention is based on the discovery of an ACCase gene (afACC1) in the fungus Aspergillus fumigatus, which is essential for survival. Essential genes are genes which are required for growth (such as metabolism, division, or reproduction) and survival of an organism. Essential genes can be used to identify therapeutic antifungal agents. These therapeutic agents can reduce or prevent growth, or decrease pathogenicity or virulence, and preferably, kill the organism.
The A. fumigatus ACCase (afACCase) coding sequence is depicted in FIGS. 1A-C as SEQ ID NO:1, and the amino acid sequence is depicted in FIG. 2 as SEQ ID NO:2. Thus the present invention relates to a novel ACCase enzymexe2x80x94which is specific to A. fumigatusxe2x80x94and to a nucleotide sequence (afACC1) encoding same. The present invention also relates to the use of the novel nucleic acid and amino acid sequences in the diagnosis and treatment of disease. The present invention also relates to the use of the novel nucleic acid and amino acid sequences to evaluate and/or to screen for agents that can modulate ACCase activity. The present invention further relates to genetically engineered host cells that include or express the novel nucleic acid and amino acid sequences to evaluate and/or to screen for agents that can modulate ACCase activity.
The ACCase enzyme of the present invention is obtainable from the A. fumigatus fungal species. This ACCase enzyme is distinguishable from the Acetyl-CoA carboxylase enzymes identified in human skeletal and adipose tissue and the yeast (S. cerevisiae) Acetyl-CoA carboxylase known as Acc1p.
The ACCase enzyme of the present invention may be the same as the naturally occurring formxe2x80x94for this aspect, e.g., the ACCase can be the non-native amino acid sequencexe2x80x94or a variant, homolog, fragment or derivative thereof. In addition, or in the alternative, the ACCase is isolated ACCase and/or purified ACCase. The ACCase can be obtainable from or produced by any suitable source, whether natural or not, or it may be synthetic, semi-synthetic or recombinant.
The ACCase gene of the invention is essential for survival of A. fumigatus. Accordingly, the ACCase nucleic acid sequence of the invention, and the ACCase polypeptide of the invention, are useful targets for identifying compounds that are inhibitors of A. fumigatus. Such inhibitors attenuate fungal growth by inhibiting the activity of the ACCase polypeptide, or by inhibiting transcription or translation. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding A. fumigatus ACCase polypeptides or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of ACCase-encoding nucleic acids (e.g., fragments of at least 15 nucleotides (e.g., at least 18, 20, or 25 nucleotides)).
The invention features a nucleic acid molecule that is at least 65% (or 75%, 85%, 95%, 98%, or 100%) identical to the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence of the cDNA insert of the plasmid deposited with the ATCC (10801 University Blvd., Manassas, Va. 209110-2209, USA) on Dec. 15, 1998 as Accession Number 207005, 207006, 207007, 207008, or 207009 (the xe2x80x9ccDNA of ATCC 207005, 207006, 207007, 207008, or 207009xe2x80x9d), or a complement thereof. The deposited biological samples contain E. coli cells containing the plasmid EpAFACC-1, EpAFACC-2, EpAFACC-3, EpAFACC-4, and EpAFACC-5, respectively. Each EpAFACC plasmid contains a partial cDNA sequence of A. fumigatus ACCase, with the five plasmids together providing a complete cDNA sequence of A. fumigatus ACCase.
The invention features a nucleic acid molecule that includes a fragment of at least 300 (e.g., 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1200, 1400, 1600, or 1770) nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence of the cDNA ATCC 207005, 207006, 207007, 207008, or 207009, or a complement thereof.
The invention also features a nucleic acid molecule that includes a nucleotide sequence encoding a protein having an amino acid sequence that is at least 65% (or 75%, 85%, 95%, 98%, or 100%) identical to the amino acid sequence of SEQ ID NO:2 or the amino acid sequence encoded by the cDNA of ATCC 207005, 207006, 207007, 207008, or 207009.
Also within the invention is a nucleic acid molecule that encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, the fragment including at least 15 (25, 30, 50, 100, 150, 300, 400, or 450) contiguous amino acids of SEQ ID NO:2 or the polypeptide encoded by the cDNA of ATCC Accession Number 207005, 207006, 207007, 207008, or 207009.
In other embodiments, the invention features an isolated ACCase protein having an amino acid sequence that is at least about 65% (e.g., 75%, 85%, 95%, 98%, or 100%) identical to the amino acid sequence of SEQ ID NO:2; and an isolated ACCase protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65% (e.g., 75%, 85%, 95%, or 100%) identical to SEQ ID NO:1 or the cDNA of ATCC 207005, 207006, 207007, 207008, or 207009; and an isolated ACCase protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or the non-coding strand of the cDNA of ATCC 207005, 207006, 207007, 207008, or 207009.
Another embodiment of the invention features ACCase nucleic acid molecules that specifically detect A. fumigatus ACCase nucleic acid molecules relative to nucleic acid molecules encoding other ACCases. For example, in one embodiment, an A. fumigatus ACCase nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule that includes the nucleotide sequence of SEQ ID NO:1, or the cDNA of ATCC 207005, 207006, 207007, 207008, or 207009, or a complement thereof. In another embodiment, the A. fumigatus ACCase nucleic acid molecule is at least 300 (e.g., 400, 500, 700, 900, 1100, or 1300) nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule that includes the nucleotide sequence shown in SEQ ID NO:1, the cDNA of ATCC 207005, 207006, 207007, 207008, or 207009, or a complement thereof. In another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of an A. fumigatus ACCase nucleic acid.
Another aspect of the invention provides a vector, e.g., a recombinant expression vector, that includes an ACCase nucleic acid molecule of the invention. In another embodiment the invention provides a host cell containing such a vector. The invention also provides a method for producing ACCase protein by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector such that an ACCase protein is produced.
Another aspect of this invention features isolated or recombinant ACCase proteins and polypeptides. Typical ACCase proteins and polypeptides possess at least one biological activity possessed by naturally occurring A. fumigatus ACCase, e.g., an ability to synthesize malonyl CoA from acetyl CoA. It is not necessary that the ACCase polypeptide have activity that is equivalent to that of the wild-type A. fumigatus ACCase. For example, the ACCase polypeptide can have 20, 50, 75, 90, 100, or an even higher percent of the wild-type activity.
Since the A. fumigatus ACCase gene, which is essential for survival, has been identified, nucleic acids encoding A. fumigatus ACCase and A. fumigatus ACCase proteins can be used to identify antifungal agents. Such antifungal agents can be identified with high throughput assays to detect inhibition of ACCase activity. For example, this inhibition can be caused by small molecules binding directly to the ACCase polypeptide or by binding of small molecules to other essential polypeptides in a biochemical pathway in which ACCase participates.
The invention also provides methods of identifying agents (such as compounds, other substances, or compositions) that affect, or selectively affect, (such as inhibit or otherwise modify) the activity of and/or expression of the ACCase, by contacting the ACCase or the nucleotide sequence coding for same with the agent and then measuring the activity of the ACCase and/or the expression thereof. In a related aspect, the invention features a method of identifying agents (such as compounds, other substances or compositions comprising same) that affect (such as inhibit or otherwise modify) the activity of and/or expression of afACCase, by measuring the activity of and/or expression of afACCase in the presence of the agent or after the addition of the agent in: (a) a cell line into which has been incorporated a recombinant construct including the nucleotide sequence of the afACCase gene (e.g., SEQ ID NO:1) or an allelic variation thereof, or (b) a cell population or cell line that naturally selectively expresses afACCase, and then measuring the activity of afACCase and/or the expression thereof.
Since the Aspergillus fumigatus ACCase gene has been identified, it can be cloned into various host cells (e.g., fungi, E. coli or yeast) for carrying out such assays in whole cells). Similarly, conventional in vitro assays of ACCase activity can be used with the ACCase of the invention.
In one embodiment, the invention features a method for identifying a compound for the treatment of a fungal infection, wherein the method entails, in sequence, (i) preparing a first cell and a second cell, the first and second cells being capable of expressing afACCase, (ii) contacting the first cell with a test compound, (iii) determining the level of expression of afACCase in the first and second cells, (iv) comparing the level of expression in the first cell with the second cell, and (v) selecting the test compound for treatment of a fungal infection where expression of afACCase in the first cell is less than expression of the essential gene in the second cell, and wherein the afACCase gene is a first nucleic acid molecule which encodes a polypeptide including the amino acid sequence of SEQ ID NO: 2, or a naturally occurring allelic variant thereof, and wherein the first nucleic acid molecule hybridizes under stringent conditions to a second nucleic acid molecule, the second nucleic acid molecule consisting of a nucleotide sequence of SEQ ID NO: 1. The determination of the level of expression of the afACCase gene can be made by measuring the amount of mRNA transcribed from the afACCase gene. Alternatively, the level of afACCase encoded by the afACCase gene can be measured.
The test compound can be a small organic molecule. Alternatively, the test compound can be a test polypeptide (e.g., a polypeptide having a random or predetermined amino acid sequence; or a naturally-occurring or synthetic olypeptide) or a nucleic acid, such as a DNA or RNA molecule. The test compound can be a naturally-occurring compound or it can be synthetically produced. Synthetic libraries, chemical libraries, and the like can be screened to identify compounds that bind to ACCase.
In another suitable method, there is provided an assay method for identifying an agent that can affect Acetyl CoA Carboxylase (ACC) activity or expression thereof, the assay method comprising contacting an agent with an amino acid sequence according to the present invention or a nucleotide sequence according to the present invention; and measuring the activity or expression of ACC; wherein a difference in activity between a) ACCase activity or expression in the absence of the agent and b) ACCase activity or expression in the presence of the agent is indicative that the agent can affect ACCase activity or expression.
Another suitable method for identifying antifungal compounds involves screening for small molecules that specifically bind to ACCase. A variety of suitable binding assays are known in the art as described, for example, in U.S. Pat. Nos. 5,585,277 and 5,679,582, hereby incorporated herein by reference. For example, in various conventional assays, test compounds can be assayed for their ability to bind a polypeptide by measuring the ability of the small molecule to stabilize the polypeptide in its folded, rather than unfolded, state. More specifically, one can measure the degree of protection against unfolding that is afforded by the test compound. Test compounds that bind afACCase with high affinity cause, for example, a significant shift in the temperature at which the polypeptide is denatured. Test compounds that stabilize the polypeptide in a folded state can be further tested for antifungal activity in a standard susceptibility assay.
In a related method for identifying antifungal compounds, an ACCase polypeptide is used to isolate peptide or nucleic acid ligands that specifically bind to the ACCase polypeptides. These peptide or nucleic acid ligands are then used in a displacement screen to identify small molecules that bind to the ACCase polypeptide. Such binding assays can be carried out as described herein.
The A. fumigatus ACCase polypeptides also can be used in assays to identify test compounds that bind to the polypeptides. Test compounds that bind to the ACCase polypeptides then can be tested, in conventional assays, for their ability to inhibit fungal growth. Test compounds that bind to the ACCase polypeptides are candidate antifungal agents, in contrast to compounds that do not bind to the ACCase polypeptides. As described herein, any of a variety of art-known methods can be used to assay for binding of test compounds to the ACCase polypeptides.
The invention includes, for example, a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) measuring the level of expression of the afACCase gene in a cell in the presence of a test compound; (b) comparing the level of expression measured in step (a) to the level of expression of the afACCase gene in a cell in the absence of the test compound; and (c) selecting the test compound as being useful for treating a fungal infection when the level of expression of the afACCase gene in the presence of the test compound is less than the level expression of the afACCase gene in the absence of the test compound, and wherein the afACCase gene has the sequence of SEQ ID NO: 1. If desired, the level of expression can be measured by measuring the amount of mRNA from the afACCase gene described herein, or by measuring the amount of protein encoded by the afACCase gene described herein. Typically, the cell is A. fumigatus or Saccharomyces (e.g., Saccharomyces cerevisiae).
In a variation of the above method, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) measuring the activity of the afACCase gene in a cell in the presence of a test compound; (b) comparing the activity measured in step (a) to the level activity of the afACCase gene in a cell in the absence of the test compound; and (c) selecting the test compound as being useful for treating fungal infections when the level of activity of the afACCase gene measured in the presence of the test compound is less than the level of activity of the afACCase gene measured in the absence of the test compound, wherein the afACCase gene has the sequence of SEQ ID NO: 1.
In an alternative method, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) measuring, in the presence of a test compound, the growth of a sample of cells which have been engineered to express a afACCase gene; (b) comparing the growth measured in step (a) to the growth of a sample of the cells in the absence of the test compound; and (c) selecting the test compound as being useful for treating a fungal infection when the growth of the sample of cells in the presence of the test compound is slower than the growth of a sample of cells in the absence of the test compound, wherein the afACCase gene has the sequence of SEQ ID NO: 1. Typically, the cell sample contains fungal cells (e.g., A. fumigatus).
The invention also includes a method for identifying an antifungal agent where the method entails: (a) contacting an ACCase polypeptide with a test compound; (b) detecting binding of the test compound to the polypeptide; and (c) determining whether a test compound that binds to the polypeptide inhibits growth of A. fumigatus, relative to growth of fungi cultured in the absence of the test compound, as an indication that the test compound is an antifungal agent. If desired, the test compound can be immobilized on a substrate, and binding of the test compound to afACCase is detected as immobilization of afACCase on the immobilized test compound. Immobilization of afACCase on the test compound can be detected in an immunoassay with an antibody that specifically binds to afACCase.
In still another method, binding of a test compound to an ACCase polypeptide can be detected in a conventional two-hybrid system for detecting protein/protein interactions (e.g., in yeast or mammalian cells). A test compound found to bind to afACCase can be further tested for antifungal activity in a conventional susceptibility assay. Generally, in such two-hybrid methods, (a) afACCase is provided as a fusion protein that includes the polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; (b) the test polypeptide is provided as a fusion protein that includes the test polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor; and (c) binding of the test polypeptide to the polypeptide is detected as reconstitution of a transcription factor. Reconstitution of the transcription factor can be detected, for example, by detecting transcription of a gene that is operably linked to a DNA sequence bound by the DNA-binding domain of the reconstituted transcription factor (See, for example, White, 1996, Proc. Natl. Acad. Sci. 93:10001-10003 and references cited therein and Vidal et al., 1996, Proc. Natl. Acad. Sci. 93:10315-10320).
In an alternative method, an isolated nucleic acid molecule encoding an ACCase is used to identify a compound that decreases the expression of ACCase in vivo (i.e., in an A. fumigatus cell). Such compounds can be used as antifungal agents. To discover such compounds, cells that express an ACCase are cultured, exposed to a test compound (or a mixture of test compounds), and the level of ACCase expression or activity is compared with the level of ACCase expression or activity in cells that are otherwise identical but that have not been exposed to the test compound(s). Standard quantitative assays of gene expression and ACCase activity can be utilized in this aspect of the invention.
To identify compounds that modulate expression of ACCase the test compound(s) can be added at varying concentrations to the culture medium of A. fumigatus. Such test compounds can include small molecules (typically, non-protein, non-polysaccharide chemical entities), polypeptides, and nucleic acids. The expression of ACCase is then measured, for example, by Northern blot PCR analysis or RNAse protection analyses using a nucleic acid molecule of the invention as a probe. The level of expression in the presence of the test molecule, compared with the level of expression in its absence, will indicate whether or not the test molecule alters the expression of afACCase. Because ACCase is essential for survival, test compounds that inhibit the expression and/or function of ACCase will inhibit growth of, or kill, the cells that express ACCase.
More generally, binding of a test compound to an ACCase polypeptide can be detected either in vitro or in vivo. If desired, the above-described methods for identifying compounds that modulate the expression of the ACCase polypeptides of the invention can be combined with measuring the levels of ACCase expressed in cells, e.g., by carrying out an assay of ACCase activity, as described above or, for example, performing a Western blot analysis using antibodies that bind to ACCase. The antifungal agents identified by the methods of the invention can be used to inhibit a wide spectrum of pathogenic or non-pathogenic fungal strains.
The invention also features a method for identifying an antifungal agent, where the method entails (a) contacting an afACCase polypeptide with a test compound; (b) detecting a decrease in activity of afACCase the contacted with test compound; (c) selecting a compound useful for treating a fungal infection as one that decreases the activity of afACCase; and, optionally, (d) determining whether a test compound that decreases activity of a contacted afACCase polypeptide inhibits growth of fungi, relative to growth of fungi cultured in the absence of a test compound that decreases activity of a contacted ACCase polypeptide, wherein inhibition of growth indicates that the test compound is an antifungal agent, and wherein afACCase is encoded by a gene having the sequence of SEQ ID NO: 1. The test compound can be, without limitation, a polypeptide, ribonucleic acid, small molecule, deoxyribonucleic acid, antisense oligonucleotide, or ribozyme.
In yet another embodiment, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) contacting a variant, homolog, or ortholog of an ACCase polypeptide with a test compound; (b) detecting binding of the test compound to the variant, homolog, or ortholog of afACCase; and (c) selecting a compound useful for treating a fungal infection as one that binds to the variant, homolog, or ortholog of afACCase, wherein afACCase is encoded by a gene having the sequence of SEQ ID NO: 1. Optionally, the method can also include (d) determining whether a test compound that binds to the variant, homolog, or ortholog of afACCase inhibits growth of fungi, relative to growth of fungi cultured in the absence of a test compound that binds to the variant, homolog, or ortholog of afACCase, wherein inhibition of growth indicates that the test compound is an antifungal agent. The variant, homolog, or ortholog can be derived from a non-pathogenic, or pathogenic, fungus.
Some specific embodiments of the present invention relate to assay methods for the identification of antifungal agents using assays for antifungal agents which may be carried out both in whole cell preparations and in ex vivo cell-free systems. In each instance, the assay target is the ACCase nucleotide sequencexe2x80x94which is essential for fungal viabilityxe2x80x94and/or the ACCase polypeptide. Candidate agents which are found to inhibit the target nucleotide sequence and/or afACCase in any assay method of the present invention are thus identified as potential antifungal agents. It is expected that the assay methods of the present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays such as serial dilution studies wherein the target ACCase nucleotide sequence or the ACCase polypeptide are exposed to a range of candidate agent concentrations.
When the assay methods of the present invention are carried out as a whole-cell assay, the target ACCase nucleotide sequence and/or the ACCase polypeptide and the entire living fungal cell may be exposed to the candidate agent under conditions normally suitable for growth. Optimal conditions including essential nutrients, optimal temperatures and other parameters, depend upon the particular fungal strain being used and suitable conditions are well known in the art. Inhibition of expression of the target nucleotide sequence and/or the activity of afACCase may be determined in a number of ways including observing the cell culture""s growth or lack thereof. Such observation may be made visually, by optical densitometric or other light absorption/scattering means or by yet other suitable means, whether manual or automated.
In the above whole-cell assay, an observed lack of cell growth may be due to inhibition of the target nucleotide sequence and/or afACCase or may be due to an entirely different effect of the candidate agent, and further evaluation may be required to establish the mechanism of action and to determine whether the candidate agent is a specific inhibitor of the target. Accordingly, and in one embodiment of the present invention, the method may be performed as a paired-cell assay in which each test compound is separately tested against two different fungal cells, the first fungal cells having a target with altered properties making it more susceptible to inhibition compared with that of the second fungal cells.
One manner of achieving differential susceptibility is by using mutant strains expressing a modified target ACCase polypeptide. A particularly useful strain is one having a temperature sensitive (xe2x80x9ctsxe2x80x9d) mutation as a result of which the target is more prone than the wild type target to loss of functionality at high temperatures (that is, temperatures higher than optimal, but still permitting growth in wild type cells). When grown at semi-permissive temperatures, the activity of a ts mutant target may be attenuated but sufficient for growth.
Alternatively or in conjunction with the above, differential susceptibility to target inhibitors may be obtained by using a second fungal cell which has altered properties making it less susceptible to inhibition compared with that of wild type cells as for example, a fungal cell which has been genetically manipulated to cause overexpression of the target. Such overexpression can be achieved by placing into a wild type cell a plasmid carrying the nucleotide sequence for the target. The techniques for generating temperature sensitive mutants, for preparing specific plasmids and for transforming cell lines with such plasmids are well known in the art.
Alternatively or in conjunction with the above, the access of candidate agents to a cell or an organism, may be enhanced by mutating or deleting a gene or genes which encode a protein or proteins responsible for providing a permeability barrier for a cell or an organism.
The present invention also relates to a method for identifying antifungal agents utilizing fungal cell systems that are sensitive to perturbation to one or several transcriptional/translational components.
By way of example, the present invention relates to a method of constructing mutant fungal cells in which one or more of the transcriptional/translational components is present in an altered form or in a different amount compared with a corresponding wild-type cell. This method further involves examining a candidate agent for its ability to perturb transcription/translation by assessing the impact it has on the growth of the mutant and wild-type cells. Agents that perturb transcription/translation by acting on a particular component that participates in transcription/translation may cause a mutant fungal cell which has an altered form or amount of that component to grow differently from the corresponding wild-type cell, but do not affect the growth relative to the wild type cell of other mutant cells bearing alterations in other components participating in transcription/translation. This method thus provides not only a means to identify whether a candidate agent perturbs transcription/translation but also an indication of the site at which it exerts its effects. The transcriptional/translational component which is present in altered form or amount in a cell whose growth is affected by a candidate agent is likely to be the site of action of the agent.
By way of example, the present invention provides a method for identifying antifungal agents which interfere with steps in translational accuracy, such as maintaining a proper reading frame during translation and terminating translation at a stop codon. This method involves constructing mutant fungal cells in which a detectable reporter polypeptide can only be produced if the normal process of staying in one reading frame or of terminating translation at a stop codon has been disrupted. This method further involves contacting the mutant fungal cells with a candidate agent to examine whether it increases or decreases the production of the reporter polypeptide.
The present invention also provides a method of screening an agent for specific binding affinity with afACCase (or a derivative, homolog, variant or fragment thereof) or the nucleotide sequence coding for same (including a derivative, homolog, variant or fragment thereof), the method comprising the steps of: a) providing a candidate agent; b) combining afACCase (or the derivative, homolog, variant or fragment thereof) or the nucleotide sequence coding for same (or the derivative, homolog, variant or fragment thereof) with the candidate agent for a time sufficient to allow binding under suitable conditions; such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the ACCase polypeptide or the nucleotide sequence encoding same with the agent; and c) determining whether the agent binds to or otherwise interacts with and activates or inhibits an activity of afACCase (or the derivative, homolog, variant or fragment thereof) or the expression of the nucleotide sequence coding for same (or the derivative, homolog, variant or fragment thereof) by detecting the presence or absence of a signal generated from the binding and/or interaction of the agent with afACCase (or the derivative, homolog, variant or fragment thereof) or the nucleotide sequence coding for same (or the derivative, homolog, variant or fragment thereof).
In other embodiments, the cell system is an extract of a fungal cell that is grown under defined conditions, and the method involves measuring transcription or translation in vitro. Such defined conditions are selected so that transcription or translation of the reporter is increased or decreased by the addition of a transcription inhibitor or a translation inhibitor to the cell extract.
One such method for identifying antifungal agents relies upon a transcription-responsive gene product. This method involves constructing a fungal cell in which the production of a reporter molecule, measured as a percentage of over-all transcription, increases or decreases under conditions in which overall fungal cell transcription is reduced. Specifically, the reporter molecule is encoded by a nucleic acid transcriptionally linked to a sequence constructed and arranged to cause a relative increase or decrease in the production of the reporter molecule when overall transcription is reduced. Typically, the overall transcription is measured by the expression of a second indicator gene whose expression, when measured as a percentage of overall transcription, remains constant when the overall transcription is reduced. The method further involves contacting the fungal cell with a candidate agent, and determining whether the agent increases or decreases the production of the first reporter molecule in the fungal cell.
In one embodiment, the reporter molecule is itself the transcription-responsive gene product whose production increases or decreases when overall transcription is reduced. In another embodiment, the reporter is a different molecule whose production is linked to that of the transcription-responsive gene product. Such linkage between the reporter and the transcription-responsive gene product can be achieved in several ways. A gene sequence encoding the reporter may, for example, be fused to part or all of the gene encoding the transcription-responsive gene product and/or to part or all of the genetic elements which control the production of the gene product. Alternatively, the transcription-responsive gene product may stimulate transcription of the gene encoding the reporter, either directly or indirectly.
Alternatively, the method for identifying antifungal agents relies upon a translation-responsive gene product. This method involves constructing a fungal cell in which the production of a reporter molecule, measured as a percentage of over-all translation, increases or decreases under conditions in which overall fungal cell translation is reduced. Specifically, the reporter molecule is encoded by nucleic acid either translationally linked or transcriptionally linked to a sequence constructed and arranged to cause a relative increase or decrease in the production of the reporter molecule when overall translation is reduced. Typically, the overall translation is measured by the expression of a second indicator gene whose expression, when measured as a percentage of overall translation, remains constant when the overall translation is reduced. The method further involves contacting the fungal cell with a candidate agent, and determining whether the agent increases or decreases the production of the first reporter molecule in the fungal cell.
In one embodiment, the reporter molecule is itself the translation-responsive gene product whose production increases or decreases when overall translation is reduced. In another embodiment, the reporter is a different molecule whose production is linked to that of the translation-responsive gene product. Such linkage between the reporter and the translation-responsive gene product can be achieved in several ways. A gene sequence encoding the reporter may, for example, be fused to part or all of the gene encoding the translation-responsive gene product and/or to part or all of the genetic elements which control the production of the gene product. Alternatively, the translation-responsive gene product may stimulate translation of the gene encoding the reporter, either directly or indirectly.
Generally, a wide variety of reporters may be used, with typical reporters providing conveniently detectable signals (eg. by spectroscopy). By way of example, a reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.
Examples of reporter molecules include but are not limited to -galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, beta-glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabeled or fluorescent tag-labeled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes. For example, the production of the reporter molecule can be measured by the enzymatic activity of the reporter gene product, such as -galactosidase.
In another embodiment of the present invention, a selection of hybridization probes corresponding to a predetermined population of genes of the selected fungal organism may be used to specifically detect changes in gene transcription which result from exposing the selected organism or cells thereof to a candidate agent. In this embodiment, one or more cells derived from the organism is exposed to the candidate agent in vivo or ex vivo under conditions wherein the agent effects a change in gene transcription in the cell to maintain homeostasis. Thereafter, the gene transcripts, primarily mRNA, of the cell or cells are isolated by conventional means. The isolated transcripts or cDNAs complementary thereto are then contacted with an ordered matrix of hybridization probes, each probe being specific for a different one of the transcripts, under conditions wherein each of the transcripts hybridizes with a corresponding one of the probes to form hybridization pairs. The ordered matrix of probes provides, in aggregate, complements for an ensemble of genes of the organism sufficient to model the transcriptional responsiveness of the organism to a candidate agent. The probes are generally immobilized and arrayed onto a solid substrate such as a microtiter plate. Specific hybridization may be effected, for example, by washing the hybridized matrix with excess non-specific oligonucleotides. A hybridization signal is then detected at each hybridization pair to obtain a transcription signal profile. A wide variety of hybridization signals may be used. In one embodiment, the cells are pre-labeled with radionucleotides such that the gene transcripts provide a radioactive signal that can be detected in the hybridization pairs. The transcription signal profile of the agent-treated cells is then compared with a transcription signal profile of negative control cells to obtain a specific transcription response profile to the candidate agent.
A variety of protocols for detecting and measuring the expression of afACCase, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on afACCase polypeptides is suitable; alternatively, a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al. (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul Minn.) and Maddox D E et al. (1983, J. Exp. Med. 15 8:121).
In an embodiment of the present invention, afACCase or a variant, homolog, fragment or derivative thereof and/or a cell line that expresses afACCase or variant, homolog, fragment or derivative thereof may be used to screen for antibodies, peptides, or other agents, such as organic or inorganic molecules, that act as modulators of afACCase activity, thereby identifying a therapeutic agent capable of modulating the activity of afACCase. For example, antibodies that specifically bind an ACCase polypeptide and are capable of neutralizing the activity of afACCase may be used to inhibit afACCase activity. Alternatively, screening of peptide libraries or organic libraries made by combinatorial chemistry with recombinantly expressed ACCase polypeptide or a variant, homolog, fragment or derivative thereof or cell lines expressing afACCase or a variant, homolog, fragment or derivative thereof may be useful for identification of therapeutic agents that function by modulating afACCase activity. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art. For example, nucleotide sequences encoding the N-terminal region of afACCase can be expressed in a cell line and used for screening of allosteric modulators, either agonists or antagonists, of afACCase activity.
Accordingly, the present invention provides a method for screening a plurality of agents for specific binding affinity with afACCase, or a portion, variant, homolog, fragment or derivative thereof, by providing a plurality of agents; combining afACCase or a portion, variant, homolog, fragment or derivative thereof with each of a plurality of agents for a time sufficient to allow binding under suitable conditions; and detecting binding of afACCase, or portion, variant, homolog, fragment or derivative thereof, to each of the plurality of agents, thereby identifying the agent or agents which specifically bind afACCase. In such an assay, the plurality of agents may be produced by combinatorial chemistry techniques known to those of skill in the art.
Another technique for screening provides for high throughput screening of agents having suitable binding affinity to afACCase polypeptides and is based upon the method described in detail in WO 84/03564. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test agents are reacted with afACCase fragments and washed. A bound ACCase polypeptide is then detectedxe2x80x94such as by appropriately adapting methods well known in the art. A purified ACCase polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
Typically, in an antifungal discovery process, potential new antifungal agents are tested for their ability to inhibit the in vitro activity of the purified expression product of the present invention in a biochemical assay. Agents with inhibitory activity can then progress to an in vitro antifungal activity screening using a standard MIC (Minimum Inhibitory Concentration) test (based on the M27-A NCCLS approved method). Antifungal active agents identified at this point are then tested for antifungal efficacy in vivo, such as by using rodent systemic candidiasis/aspergillosis models. Efficacy is measured by measuring the agent""s ability to increase the host animal""s survival rate against systemic infection, and/or reduce the fungal burden in infected tissues, compared to control animals receiving no administered agent (which can be by oral or intravenous routes).
The present invention also provides a pharmaceutical composition for treating an individual in need of such treatment of a disease caused by afACCase activity (or that can be treated by inhibiting afACCase activity); the treatment method entails administering a therapeutically effective amount of an agent that affects (such as inhibits) the activity and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
The pharmaceutical compositions can be used for humans or animals and will typically include any one or more of a pharmaceutically acceptable diluent, carrier, excipient or adjuvant. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions can include as (or in addition to) the carrier, excipient, or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s).
The invention includes pharmaceutical formulations that include a pharmaceutically acceptable excipient and an antifungal agent identified using the methods described herein. In particular, the invention includes pharmaceutical formulations that contain antifungal agents that inhibit the growth of, or kill, pathogenic fungal strains (e.g., pathogenic Aspergillus fumigatus strains). Such pharmaceutical formulations can be used in a method of treating a fungal infection in an organism. Such a method entails administering to the organism a therapeutically effective amount of the pharmaceutical formulation, i.e., an amount sufficient to ameliorate signs and/or symptoms of the fungal infection. In particular, such pharmaceutical formulations can be used to treat fungal infections in mammals such as humans and domesticated mammals (e.g., cows, pigs, dogs, and cats), and in plants. The efficacy of such antifungal agents in humans can be estimated in an animal model system well known to those of skill in the art (e.g., mouse systems of fungal infections).
The invention also includes (i) a method of treating a mycosal and/or fungal infection in a target (which target can be a living organism, such as a mammal, such as a human, or an inanimate target, such as a textile piece, paper, plastic etc.), which method entails delivering (such as administering or exposing) an effective amount of an agent capable of modulating the expression pattern of the nucleotide sequence of the present invention or the activity of the expression product thereof; and (ii) a method of treating a mycosal and/or fungal infection in a target (which target can be a living organism, such as a plant or a mammal, such as a human, or an inanimate target, such as a textile piece, paper, plastic etc.), which method entails delivering (such as administering or exposing) an effective amount of an agent identified by an assay according to the present invention. As used herein, the terms xe2x80x9ctreating,xe2x80x9d xe2x80x9ctreat,xe2x80x9d or xe2x80x9ctreatmentxe2x80x9d include, inter alia, preventative (e.g., prophylactic), palliative, and curative treatment of fungal infections.
The invention also features a method for inducing an immunological response in an individual, particularly a mammal, which entails inoculating the individual with one or more of the ACCase genes or polypeptides described herein, and generally in an amount adequate to produce an antibody and/or T cell immune response to protect the individual from mycoses, fungal infection, or infestations. In another aspect, the present invention relates to a method of inducing an immunological response in an individual which entails delivering to the individual a vector that includes an ACCase gene described herein or a variant, homolog, fragment, or derivative thereof in vivo to induce an immunological response, such as to produce antibody and/or a T-cell immune response to protect the individual from disease whether that disease is already established within the individual or not.
Various affinity reagents that are permeable to the microbial membrane (i.e., antibodies and antibody fragments) are useful in practicing the methods of the invention. For example polyclonal and monoclonal antibodies that specifically bind to the A. fumigatus ACCase polypeptide can facilitate detection of A. fumigatus ACCase in various fungal strains (or extracts thereof). These antibodies also are useful for detecting binding of a test compound to ACCase (e.g., using the assays described herein). In addition, monoclonal antibodies that specifically bind to A. fumigatus ACCase can themselves be used as antifungal agents.
In another aspect, the invention features a method for detecting an A. fumigatus ACCase polypeptide in a sample. This method includes: obtaining a sample suspected of containing an A. fumigatus ACCase polypeptide; contacting the sample with an antibody that specifically binds to an A. fumigatus ACCase polypeptide under conditions that allow the formation of complexes of the antibody and the ACCase polypeptide; and detecting the complexes, if any, as an indication of the presence of an A. fumigatus ACCase polypeptide in the sample.
In all of the foregoing methods, homologs, orthologs, or variants of the ACCase genes and polypeptides described herein can be substituted. While xe2x80x9chomologsxe2x80x9d are structurally similar genes contained within a species, xe2x80x9corthologsxe2x80x9d are functionally equivalent genes from other species (within or outside of a given genus, e.g., from E. coli). The terms xe2x80x9cvariant,xe2x80x9d xe2x80x9chomolog,xe2x80x9d or xe2x80x9cfragmentxe2x80x9d in relation to the amino acid sequence of the ACCase of the invention include any substitution, variation, modification, replacement, deletion, or addition of one or more amino acids from or to the sequence providing the resultant ACCase polypeptide.
The invention offers several advantages. The invention provides targets, based on essential functions, for identifying potential agents for the effective treatment of opportunistic infections caused by A. fumigatus and other related fungal species. Also, the methods for identifying antifungal agents can be configured for high throughput screening of numerous candidate antifungal agents. Because the ACCase gene disclosed herein is thought to be highly conserved, antifungal drugs targeted to this gene or its gene products are expected to have a broad spectrum of antifungal activity.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and are not intended to limit the scope of the invention, which is defined by the claims.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.